Table_3_RNA-seq analysis revealed considerable genetic diversity and enabled the development of specific KASP markers for Psathyrostachys huashanica.xlsx

Psathyrostachys huashanica, which grows exclusively in Huashan, China, is an important wild relative of common wheat that has many desirable traits relevant for wheat breeding. However, the poorly characterized interspecific phylogeny and genomic variations and the relative lack of species-specific molecular markers have limited the utility of P. huashanica as a genetic resource for enhancing wheat germplasm. In this study, we sequenced the P. huashanica transcriptome, resulting in 50,337,570 clean reads that were assembled into 65,617 unigenes, of which 38,428 (58.56%) matched at least one sequence in public databases. The phylogenetic analysis of P. huashanica, Triticeae species, and Poaceae species was conducted using 68 putative orthologous gene clusters. The data revealed the distant evolutionary relationship between P. huashanica and common wheat as well as the substantial diversity between the P. huashanica genome and the wheat D genome. By comparing the transcriptomes of P. huashanica and Chinese Spring, 750,759 candidate SNPs between P. huashanica Ns genes and their common wheat orthologs were identified. Among the 90 SNPs in the exon regions with different functional annotations, 58 (64.4%) were validated as Ns genome-specific SNPs in the common wheat background by KASP genotyping assays. Marker validation analyses indicated that six specific markers can discriminate between P. huashanica and the other wheat-related species. In addition, five markers are unique to P. huashanica, P. juncea, and Leymus species, which carry the Ns genome. The Ns genome-specific markers in a wheat background were also validated regarding their specificity and stability for detecting P. huashanica chromosomes in four wheat–P. huashanica addition lines. Four and eight SNP markers were detected in wheat–P. huashanica 2Ns and 7Ns addition lines, respectively, and one marker was specific to both wheat–P. huashanica 3Ns, 4Ns, and 7Ns addition lines. These markers developed using transcriptome data may be used to elucidate the genetic relationships among Psathyrostachys, Leymus, and other closely-related species. They may also facilitate precise introgressions and the high-throughput monitoring of P. huashanica exogenous chromosomes or segments in future crop breeding programs.

华山山羊草（Psathyrostachys huashanica），一种仅生长于中国华山的特有植物，作为普通小麦的重要野生近缘种，具备众多对小麦育种有益的性状。然而，其不甚清晰的种间系统发育关系、基因组变异以及物种特异性分子标记的相对匮乏，限制了华山山羊草作为遗传资源在提升小麦种质资源方面的应用。在本研究中，我们对华山山羊草转录组进行了测序，获得了50,337,570条清洁读段，组装成65,617个非冗余基因（unigenes），其中38,428个（占比58.56%）至少与公共数据库中的一个序列相匹配。通过68个假定的直系同源基因簇，对华山山羊草、麦属（Triticeae）物种和禾本科（Poaceae）物种进行了系统发育分析。数据揭示了华山山羊草与普通小麦之间遥远的进化关系以及华山山羊草基因组与小麦D基因组之间显著的多样性。通过比较华山山羊草与中国春的转录组，识别了华山山羊草Ns基因及其在普通小麦中的同源基因之间的750,759个候选单核苷酸多态性（SNPs）。在90个具有不同功能注释的外显子区域SNPs中，58个（占比64.4%）通过KASP基因分型检测被验证为普通小麦背景下的Ns基因组特异性SNPs。标记验证分析表明，六个特定标记可以区分华山山羊草与其他小麦相关物种。此外，五个标记仅存在于华山山羊草、芸苔（P. juncea）和赖草（Leymus）物种中，这些物种携带Ns基因组。在小麦背景下的Ns基因组特异性标记也经过验证，其在检测四个小麦-华山山羊草附加系中的华山山羊草染色体方面的特异性和稳定性。在小麦-华山山羊草2Ns和7Ns附加系中分别检测到四个和八个SNP标记，而在小麦-华山山羊草3Ns、4Ns和7Ns附加系中检测到一个特异性标记。这些基于转录组数据开发的标记可能用于阐明Psathyrostachys、Leymus和其他近缘物种之间的遗传关系，并可能促进精确的基因导入以及未来作物育种计划中华山山羊草外源染色体或片段的高通量监测。